Members of the FC receptor homolog gene family (FcRH1-3,6) related reagents and uses thereof

ABSTRACT

The invention relates to members of the Fc receptor homolog (FcRH) subfamily, as well as fragments and variants thereof. Each FcRH is a Type I transmembrane receptor, preferably, comprises an extracellular region, a transmembrane region, and a cytoplasmic region. The cytoplasmic region preferably comprises one or more immunoreceptor tyrosine-based inhibitory or activation motifs (“ITIMs” or “ITAMs). The invention provides polypeptides, nucleic acids, vectors, expression systems, and antibodies and antibody fragments related to the FcRHs as well as uses thereof. Such uses include uses in the diagnosis and treatment of a malignancy of hematopoietic cell lineage or an inflammatory or autoimmune disease in a subject and in the modulation of a humoral immune response in a subject.

CROSS-REFERENCED TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/508,374 filed Mar.25, 2005, which is a National Stage filing under 35 USC §371 ofPCT/US2003/009600 filed Mar. 25, 2003, which claims the benefit of U.S.Provisional Application No. 60/367,667, filed Mar. 25, 2002, all ofwhich are incorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grants 2R37 andAI39816 awarded by NIAID. The government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates generally to immunology and modulation ofimmunologic responses in the context of inflammatory diseases andcancer.

BACKGROUND OF THE INVENTION

Receptors for the Fc region (FcRs) of Igs have broad tissue distributionpatterns and can modulate cellular and humoral immunity by linking theirantibody ligands with effector cells of the immune system (Ravetch, J.V. & Kinet, J.-P. (1991) Annu. Rev. Immunol. 9, 457-492; Daeron, M.(1997) Annu. Rev. Immunol. 15, 203-234. These cellular receptors havethe ability to sense humoral concentrations of antibody, initiatecellular responses in host defense, and participate in autoimmunedisorders (Ravetch, J. V. & Bolland, S. (2001) Annu. Rev. Immunol. 19,275-290). Their diverse regulatory roles depend on the Ig isotypespecificity and cellular distribution of the individual FcR. These Igsuperfamily members share similarities in their ligand binding subunits,and they may have inhibitory or activating signaling motifs in theirintracellular domains or instead pair with signal transducing subunitspossessing activating signaling motifs.

Recently, characterization of FcR homologs in mice, the paired Ig-likereceptors (Kubagawa, H. et al. (1997) Proc. Natl. Acad. Sci. USA 94,5261-5266; Hayami, K. et al. (1997) J. Biol. Chem. 272, 7320-7327), andtheir relatives in humans the Ig-like transcripts/leucocyte Ig-likereceptors (Borges, L. et al. (1997) J. Immunol. 159, 5192-5196;Samaridis, J. & Colonna, M. (1997) Eur. J. Immunol. 27, 660-665) havebeen elucidated. This multigene family, which includes the FcαR (Kremer,E. J. et al. (1992) Hum. Genet. 89, 107-108) and the natural killer cellIg-like receptors (Wagtmann, N. et al. (1997) Curr. Biol. 7, 615-618),is located in a human chromosome 19q13 region known as the leucocytereceptor complex (LRC) (Wende, H. et al. (1999) Mamm. Genome 10,154-160; Wilson, M. J. et al. (2000) Proc. Natl. Acad. Sci. USA 97,4778-4783). These Ig-like multigene families belong to a larger class ofreceptors characterized by their possession of common cytoplasmictyrosine-based signaling motifs. These can be either immunoreceptortyrosine-based activation motifs (ITAMs) containing two repeats of theconsensus sequence Y-X-X-L/I spaced by 6-8 amino acids(E/D)-X-X-Y-X-X-(L/I)-X₆₋₈-Y-X-X-(L/I) (SEQ ID NO:64, with six aminoacid between the consensus sequences; SEQ ID NO:65, with seven aminoacid residues between the consensus sequences; and SEQ ID NO:66, witheight amino acid residues between the consensus sequences) orimmunoreceptor tyrosine-based inhibitory motifs (ITIMs) with a 6-aminoacid consensus sequence (I/V/L/S)-X-Y-X-X-(L/V) (SEQ ID NO:67) (Reth, M.(1992) Annu. Rev. Immunol. 10, 97-121; Vely, F. & Vivier, E. (1997) J.Immunol. 159, 2075-2077; Ravetch, J. V. & Lanier, L. L. (2000) Science290, 84-89; Gergely, J. et al. (1999) Immunol. Lett. 68, 3-15). Thephylogenetic conservation of these types of receptors in birds (Dennis,G. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 13245-13250) and bonyfish (Yoder, J. A. et al. (2001) Proc. Natl. Acad. Sci. USA 98,6771-6717) is indicative of their biological value. After ligand bindingof the activating receptor complexes, ITAM tyrosines are rapidlyphosphorylated by Src family kinases to initiate a cascade of signalingevents that trigger cellular activation. In the case of ITIM-bearingreceptors, the tyrosines provide a docking site for phosphatasescontaining Src homology 2 domains that can abrogate cellular activation(Long, E. O. (1999) Annu. Rev. Immunol. 17, 875-904; Unkeless, J. C. &Jin, J. (1997) Curr. Opin. Immunol. 9, 338-343). The balance in theutilization of these activating and inhibitory receptor pairs can serveto modulate cellular responses to a variety of stimuli.

The genes encoding the classical FcγRs, FcγRI, FcγRII, FcγRIII, andFcεRI, lie on the long arm of chromosome 1 (1q21-23) near the polymericIg receptor (pIgR) and Fcα/μR genes (1q32) (20-23). Members of this FcRsubfamily have relatively low extracellular homology with theFcR-related genes that reside in the LRC on chromosome 19. Like theFcγR- and FcεR-activating receptors, the ligand binding chain of theFcαR coassociates with the ITAM containing FcR common γ-chain(Pfefferkorn, L. C. & Yeaman, G. R. (1994) J. Immunol. 153, 3228-3236;Morton, E. C. et al. (1995) J. Biol. Chem. 270, 29781-29787). Newmembers of the FcR family were sought which could have diverse signallyproperties and oncogenic potential.

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates tomembers of a cluster of FcR and FcR gene relatives encoded, for example,by genes in the human chromosome 1q21-23 region, or analogous region innon-human subjects. The members are Type I transmembrane receptors, oralternatively spliced forms thereof, with homology to the FcR family andare referred to herein as FcRHs. Each FcRH can comprise an extracellularregion, a transmembrane region, and a cytoplasmic region. Thecytoplasmic region preferably comprises one or more immunoreceptortyrosine-based inhibitory or activation motifs (“ITIMs” or “ITAMs).

The invention relates to polypeptides corresponding to isolated FcRHs(e.g., huFcRH 1, 2, 3, and 6 and moFcRH1, 2, and 3), as well asfragments and isoforms thereof. The invention further relates to nucleicacids that encode the FcRHs, as well as hybridization probes relatedthereto and complementary sequences. The invention further providesvectors and cells related to the nucleic acids of the invention.

The invention further relates to making an FcRH, or a fragment orvariant thereof, comprising culturing a cell comprising a vector of theinvention under conditions permitting expression of the FcRH. Theinvention also provides an antibody reagent kit comprising the antibody,or a fragment or variant thereof, and reagents for detecting binding ofthe antibody, fragment, or antibody variant to a ligand.

The invention further relates to uses of the polypeptides, nucleic acidsand antibodies of the invention. For example, the invention relates tomethods of diagnosing and methods of treating a malignancy ofhematopoietic cell lineage or an inflammatory or autoimmune disease in asubject. The invention also relates to modulation of a humoral immuneresponse in a subject.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate (one) several embodiment(s) ofthe invention and together with the description, serve to explain theprinciples of the invention.

FIG. 1 shows the relative position of the FcRH locus within the FcRcluster on chromosome 1. The cytogenetic location of the FcR genes isapproximated from the GenBank Mapview database. The BAC clones (4,GenBank accession no. AL139409; 3, GenBank accession no. AL356276; 2,GenBank accession no. AL135929; and 1, GenBank accession no. AL353721)that span the locus are oriented in relation to their respective FcRHgenes (shaded area).

FIG. 2 shows the structural and sequence diversity of FcRH1, FcRH2, andFcRH3. FIG. 2A is a schematic representation of FcRH molecules. Thethree cDNAs encode type I transmembrane proteins with similarextracellular domains, but different cytoplasmic regions. Theextracellular (EC) regions contain different numbers of C2-like IGdomains and potential sites of N-linked glycosylation. The transmembrane(TM) domains are uncharged, except in the case of FcRH1. The cytoplasmic(CY) region of FcRH1 contains two ITAMs (light gray boxes) and oneITAM-like region (small, lined box), whereas FcRH2 contains one ITAM andtwo ITIMs (dark gray boxes). FcRH3 has a long cytoplasmic tail with oneITAM, one ITIM, and an ITAM-like region. The amino acid length of eachregion is indicated. FIG. 2B shows the multiple alignment comparison ofFcRH1 (SEQ ID NO:103), FcRH2 (SEQ ID NO:104), and FcRH3 (SEQ ID NO:105)amino acid sequences (one-letter code) based on the FcRH3 sequence.Amino acid identity is represented by dots, and gaps are indicated bydashes. Predicted N-linked glycosylation sites and transmembrane domainsare underlined in black. Consensus ITAM (bold) and ITIM (bold,underlined) motifs are indicated. Putative structural domains arelabeled: SP, signal peptide; EC, extracellular domain; MP-TM, membraneproximal-transmembrane; and CY, cytoplasmic regions. Amino acid lengthsare indicated in parentheses.

FIG. 3 shows a composite analysis of the extracellular homology amongFcRH and FcR family members. Pairwise analysis of individual Ig-likesubunits was performed with the CLUSTAL method algorithm using FcRH3 asthe index of comparison. Individual homologous domains are coded toindicate relatedness. Percent amino acid identities for related domainsare indicated and aligned in relation to the comparative FcRH3 subunit.The amino acid identity for the membrane proximal domains (light graysubunits) of FcRH5 are provided as the range of identity for allindividually related domains. Comparisons that are not applicable areleft blank. Amino acid sequences were derived from IRTA1 (GenBankaccession no. AF343659), IRTA2 (GenBank accession no. AF34364), moFcRH(GenBank accession no. AAG28775) FcγRI (GenBank accession no. AAA35678),FcγRII (Swiss-Prot accession no. P31994), FcγRIII (Swiss-Prot accessionno. P08637), FcεRI (Swiss-Prot accession no. P12319), and FcαRI(Swiss-Prot accession no. P24071).

FIG. 4 shows the relative location of the mouse FcR family. Location isindicated in reference to the human FcR related genes at Ch 1q21-23 andtheir orthologous loci on mouse Ch 3 and Ch 1. The microsatellite markerd3Mit187 is located within moFcRH1.

FIG. 5 shows the multiple alignment comparisons of huFcRH1-5 (SEQ IDNOs:106-110) and mouse FcRH2 and 3 (SEQ ID NOs:111 and 112) amino acidsequences (one-letter code) based on the FcRH3 sequence. Amino acid gapsare indicated by dashes. Consensus ITAM (underlined) and ITIM (italic,underlined) motifs are indicated. Amino acid lengths are indicated inparentheses.

FIG. 6 shows domains marked to indicate relatedness of the Ig-likesubunits. Ig-like domain homology was determined by generation of aphylogenetic tree using DNAStar software with the CLUSTAL program andassigning arbitrary colors to individual Ig-domains of a given branch.Amino acid identities for full length, extracellular and, cytoplasmicdomain comparisons are based on huFcRH3. Closest cytoplasmic relativesare indicated in parentheses. Most identical extracellular comparisonsbetween mouse and human relatives are highlighted in horizontal lines.Comparisons that are not applicable are left blank.

FIG. 7 shows the domains of huFcRH1-6, moFcRH1-3 and related proteins.Domains are colored to indicate relatedness of the Ig-like subunits.Ig-like domain homology was determined by generation of a phylogenetictree using DNAStar software with the CLUSTAL program and assigningarbitrary colors to individual Ig-domains of a given branch. Amino acididentities for full length, extracellular and, cytoplasmic domaincomparisons are based on huFcRH3. Closest cytoplasmic relatives areindicated in parentheses. Most identical extracellular comparisonsbetween mouse and human relatives are highlighted in red. Comparisonsthat are not applicable are left blank.

FIG. 8 shows the structural characteristics of the mouse FcRH isoforms.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein and to the Figures and their previousand following description.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to a receptor includesmixtures of various receptors, reference to “a pharmaceutical carrier”includes mixtures of two or more such carriers, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally contains two ITAMconsensus motifs” means that the two ITAMs may or may not be present andthat the description includes both the presence and absence of two ITAMconsensus motifs.

As used throughout, by “subject” is meant an individual. Preferably, thesubject is a mammal such as a primate, and, more preferably, a human.The term “subject” can include domesticated animals, such as cats, dogs,etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), andlaboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.).

By “isolated nucleic acid” is meant a nucleic acid the structure ofwhich is not identical to that of the naturally occurring nucleic acidor to that of any fragment of the naturally occurring genomic nucleicacid spanning more than three separate genes. The term therefore covers,for example, (a) a DNA which has the sequence of part of the naturallyoccurring genomic DNA molecules but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas cDNA, a genomic fragment, a fragment produced by polymerase chainreaction, or a restriction fragment; and (d) a recombinant nucleotidesequence that is part of a hybrid gene, i.e., a gene encoding a fusionprotein.

By “label” is meant any detectable tag that can be attached directly(e.g., a fluorescent molecule integrated into a polypeptide or nucleicacid) or indirectly (e.g., by way of binging to a primary antibody asecondary antibody with an integrated fluorscent molecule) to themolecule of interest. A “label” is any tag that can be visualized withimaging methods. The detectable tag can be a radio-opaque substance,radiolabel, a fluorescent label, or a magnetic label. The detectable tagcan be selected from the group consisting of gamma-emitters,beta-emitters, and alpha-emitters, gamma-emitters, positron-emitters,X-ray-emitters and fluorescence-emitters suitable for localization.Suitable fluorescent compounds include fluorescein sodium, fluoresceinisothiocyanate, phycoerythrin, and TEXAS RED® sulfonyl chloride(Molecular Probes, Eugene, Oreg.). See, de Belder & Wik (Preparation andproperties of fluorescein-labelled hyaluronate. Carbohydr. Res.44(2):251-57 (1975). Those skilled in the art will know, or will be ableto ascertain with no more than routine experimentation, otherfluorescent compounds that are suitable for labeling the molecule.

Polypeptides

The invention provides members of a cluster of FcR and FcR generelatives encoded by genes in the human chromosome 1q21-23 region, oranalogous region in non-human subjects, including for example,chromosome 3 in mouse. A consensus amino acid motif, based on the FcγRI,FcγRII, FcγRIII, and pIgR extracellular regions, was used in a GenBankprotein database query to identify member of the gene subfamily. Genomicclones were identified that were found to contain FcR relatives and aretermed the Fc receptor homolog (FcRH) subfamily: specifically, FcRH1,FcRH2, FcRH3, and FcRH6. Also, found were mouse Fc receptor homologsdesignated moFcR1, 2, and 3.

By “homologous” is meant about 25% percent homology or greater. Homologyis also characterized by proximity in the location of the genes and bysimilarities as identified in a composite analysis. As used herein,“percent homology” of two amino acid sequences or of two nucleic acidsequences is determined using the algorithm of Karlin and Altschul(Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990)). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al. (J.Mol. Biol. 215:403-410 (1990)). BLAST nucleotide searches are performedwith the NBLAST program, score 100, wordlength=12, to obtain nucleotidesequences homologous to a nucleic acid molecule of the invention. BLASTprotein searches are performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to a referencepolypeptide. To obtain gapped alignments for comparison purposes, GappedBlast is utilized as described in Altschul et al. (Nucl. Acids Res. 25:3389-3402 (1997)). When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)are used. See http://ww.ncbi.nlm.nih.gov.

By “FcRH” is meant a Type I transmembrane receptor, or an alternativelyspliced form thereof, including, for example, a secreted form or aGPI-anchored form, with homology to the classical Fc receptor family. Ina preferred embodiment, the FcRH shows homology with the extracellularregions of FcγRI, FcγRII, FcγRIII, or pIgR. More specifically, the FcRHshows homology with an amino acid sequence corresponding with the aminoterminal sequences of the second Ig domains of the FcγRs and the thirdIg domain of pIgR or FcγRH1. The FcRH can comprise an extracellularregion, a transmembrane region, and a cytoplasmic region. Theextracellular region preferably comprises one or more Ig domains, andmore preferably less than 9, and even more preferably less than 7 orless than 81 g domains. Preferably, the cytoplasmic region comprisesmore than 107 (including more than 108, 109, 110, 111, 112, 113, 114,115, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145 aminoacids). Alternatively, the cytoplasmic region comprises less than 104amino acids (including less than 103, 102, 101, 100, 99, 98, 97, 96, 95,94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80). Thecytoplasmic region preferably comprises one or more immunoreceptortyrosine-based inhibitory or activation motifs (“ITIMs” or “ITAMs).

The invention provides isolated FcRHs (e.g., huFcRH1, 2, 3, and 6, andmoFcRH1-3, as described in detail below), as well as fragments andisoforms thereof. The isolated amino acid sequences provided hereinoptionally are combined with a human signal sequence (e.g.,MLPRLLLLICAPLCEP (SEQ ID NO:29), MLLWSLLVIFDAVTEQADS (SEQ ID NO:30),MLLWLLLLLILTPGREQS (SEQ ID NO:31), MLLWTAVLLFVPCVG (SEQ ID NO:32)) or amouse signal sequence (e.g., MPLCLLLLVFAPVGVQS (SEQ ID NO:69),MLPWLLLLICALPCEPA (SEQ ID NO:72), MSGSFSPCVVFTQMWLTLLVVTPVN (SEQ IDNO:79)).

In one embodiment, the invention provides huFcRH1 and its fragments andisoforms. Thus, in one embodiment of the isolated FcRH, theextracellular region comprises less than four Ig domains. Preferably,the cytoplasmic region comprises less than 104 amino acids and, evenmore preferably, comprises less than 104 and more than 86 amino acids.In one embodiment, the transmembrane region comprises an acidic aminoacid (e.g., glutamate or aspartate). The isolated FcRH of the inventioncomprises a cytoplasmic region having the amino acid sequence of SEQ IDNO:1, in the presence or absence of conservative amino acidsubstitutions. Further provided is the isolated FcRH, wherein theextracellular region comprises the amino acid sequence of SEQ ID NO:21,in the presence or absence of conservative amino acid substitutions, andin the presence and absence of a signal sequence. More specifically, theisolated FcRH comprises the amino acid sequence of SEQ ID NO:2, in thepresence or absence of conservative amino acid substitutions, and in thepresence or absence of a signal sequence. In one embodiment the signalsequence is MLPRLLLLICAPLCEP (SEQ ID NO:29). In a preferred embodiment,the FcRH of the invention is expressed by myeloid cells (e.g.,granulocytes and monocytes). Additional characteristics of the fulllength FcRH1 include a predicted molecular weight of about 46-47kDaltons; about 425-435 (e.g., 429) amino acids in length with about 35strongly basic(+) amino acids (K,R), about 45 strongly acidic(−) aminoacids (D,E), about 144 hydrophobic amino acids (A,I,L,F,W,V), and about127 polar amino acids (N,C,Q,S,T,Y); a predicted isolelectric point ofabout 5-5.5 (e.g., 5.310); and charge of about −9 at PH 7.0.

In another embodiment, the invention provides an isolated FcRHcorresponding to huFcRH2, its fragments, or isoforms. Thus, theinvention provides a FcRH wherein the cytoplasmic region comprises lessthan 99 amino acids (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98) and wherein the receptor furthercomprises an extracellular domain with up to four Ig domains and up tofive N-linked glycosylation sites. More specifically, the isolated FcRHhas a cytoplasmic region that comprises the amino acid sequence of SEQID NO:3, in the presence or absence of conservative amino acidsubstitutions, or an extracellular region comprising SEQ ID NO:22, inthe presence or absence of conservative amino acid substitutions, and inthe presence or absence of a signal sequence. Even more specifically,the isolated FcRH comprises the amino acid sequence of SEQ ID NO:4, inthe presence or absence of conservative amino acid substitutions, and inthe presence or absence of a signal sequence. In one embodiment, thesignal sequence WSLLVIFDAVTEQADS (SEQ ID NO:30). Additionalcharacteristics of the full length FcRH2 include a predicted molecularweight of about 50-60 kDaltons; about 495-515 (e.g., 508) amino acids inlength with about 44 strongly basic(+) amino acids (K,R), about 49strongly acidic(−) amino acids (D,E), about 175 hydrophobic amino acids(A,I,L,F,W,V), and about 161 polar amino acids (N,C,Q,S,T,Y); apredicted isolelectric point of about 6-6.5 (e.g., 6.188); and charge ofabout −4 at PH 7.0.

In another embodiment, the invention provides huFcRH3, its fragments,and isoforms. More specifically, the invention provides an isolated FcRHhaving a cytoplasmic region that comprises more than 107 amino acids(e.g., 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150 amino acids). Optionally, the isolated FcRH has a cytoplasmicregion comprising one ITAM and one ITIM. More specifically, thecytoplasmic region comprises the amino acid sequence of SEQ ID NO:5 orSEQ ID NO:23, in the presence or absence of conservative amino acidsubstitutions. In one embodiment, the extracellular domain of the FcRHcomprises the amino acid sequence of SEQ ID NO:24, in the presence orabsence of conservative amino acid substitutions, and in the presence orabsence of a signal sequence. Also provided is an isolated FcRHcomprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:25, inthe presence or absence of one or more amino acid substitutions, and inthe presence or absence of a signal sequence. In one embodiment thesignal sequence comprises MLLWLLLLILTPGREQS (SEQ ID NO:31). Additionalcharacteristics of the full length FcRH3 include a predicted molecularweight of about 80-90 kDaltons; about 725-740 (e.g., 734) amino acids inlength with about 68 strongly basic(+) amino acids (K,R), about 75strongly acidic(−) amino acids (D,E), about 232 hydrophobic amino acids(A,I,L,F,W,V), and about 224 polar amino acids (N,C,Q,S,T,Y); apredicted isolelectric point of about 6.5-7.0 (e.g., 6.852); and chargeof about −2 at PH 7.0.

The invention further provides an isolated huFcRH6, its fragments, andisoforms. More specifically, the FcRH comprises a cytoplasmic regionhaving the amino acid sequence of SEQ ID NO:26, in the presence orabsence or one or more conservative amino acid substitutions. Theextracellular domain comprises the amino acid sequence of SEQ ID NO:27,in the presence or absence of conservative amino acid substitutions, andin the presence or absence of a signal sequence. Also, provided by theinvention is a FcRH having the amino acid substitutions of SEQ ID NO:28,in the presence or absence of conservative amino acid substitutions, andin the presence or absence of a signal sequence. In one embodiment thesignal sequence is MLLWTAVLLFVPCVG (SEQ ID NO:32).

The invention further provides a polypeptide comprising the amino acidsequence of SEQ ID NO:1, 21, 2, 3, 22, 4, 5, 23, 24, 6, 25, 26, 27, or28, in the presence or absence of conservative amino acid substitutions.The invention also provides a polypeptide having at least 80, 85, 90, or95% homology with SEQ ID NOs: 1, 21, 2, 3, 22, 4, 5, 23, 24, 6, 25, 26,27, or 28.

The invention further provides an isolated moFcRH1 isoform, itsfragments, and isoforms. The moFcRH1 is an isoform of SEQ ID NO:68. Morespecifically, the FcRH comprises four Ig domains, optionally having thesequence of SEQ ID NO: 70, in the presence or absence or one or moreconservative amino acid substitutions, and in the presence or absence ofa signal sequence (e.g., the sequence of SEQ ID NO:71).

The invention further provides an isolated moFcRH2, its fragments, andisoforms. The provided isoforms include one isoform with a transmembraneregion and one isoform lacking the transmembrane region. Morespecifically, the FcRH comprises a cytoplasmic region having the aminoacid sequence of SEQ ID NO:76, in the presence or absence or one or moreconservative amino acid substitutions. The extracellular domaincomprises the amino acid sequence of SEQ ID NO:74, in the presence orabsence of conservative amino acid substitutions, and in the presence orabsence of a signal sequence. Also, provided by the invention is a FcRHhaving the amino acid sequence of SEQ ID NO:73, which comprises atransmembrane region, or SEQ ID NO:77, which lacks the transmembraneregion. In each case, the FcRH sequence can include the presence orabsence of conservative amino acid substitutions, and the presence orabsence of a signal sequence. In one embodiment the signal sequence isthe sequence of SEQ ID NO:72.

The invention also provided a moFcRH3, its fragments and isoforms. Thecytoplasmic region can comprise the amino acid sequence of SEQ ID NO:81,in the presence or absence of conservative amino acid substitutions.Optionally, the extracellular domain comprises the amino acid sequenceof SEQ ID NO: 80, in the presence or absence of conservative amino acidsubstitutions or in the presence or absence of a signal sequence (e.g.,the sequence of SEQ ID NO:79). The full length sequence optionally hasthe amino acid sequence of SEQ ID NO:78, in the presence or absence ofconservative amino acid substitutions or in the presence or absence of asignal sequence (e.g., the sequence of SEQ ID NO:79).

Fragments, variants, or isoforms of the FcRHs of the invention areprovided. It is understood that these terms include functional variants.Fragments can include the cytoplasmic region, the extracellular region,the transmembrane region or any portion of at least 10 amino acids orany combination of the regions or portions. The variants are produced bymaking amino acid substitutions, deletions, and insertions, as well aspost-translational modifications. Variations in post-translationalmodifications can include variations in the type or amount ofcarbohydrate moieties of the protein core or any fragment or derivativethereof. Variations in amino acid sequence may arise naturally asallelic variations (e.g., due to genetic polymorphism) or may beproduced by human intervention (e.g., by mutagenesis of cloned DNAsequences), such as induced point, deletion, insertion and substitutionmutants. These modifications can result in changes in the amino acidsequence, provide silent mutations, modify a restriction site, orprovide other specific mutations.

Amino acid sequence modifications fall into one or more of threeclasses: substitutional, insertional or deletional variants. Insertionsinclude amino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.Deletions are characterized by the removal of one or more amino acidresidues from the protein sequence. Typically, no more than about 2 to 6residues are deleted at any one site within the protein molecule. Thesevariants ordinarily are prepared by site-specific mutagenesis ofnucleotides in the DNA encoding the protein, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known andinclude, for example, M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues but may includemultiple substitutions at different positions; insertions usually willbe on the order of about from 1 to 10 amino acid residues but can bemore; and deletions will range about from 1 to 30 residues, but can bemore. Deletions or insertions preferably are made in adjacent pairs,i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions,deletions, insertions or any combination thereof may be combined toarrive at a final construct. The mutations must not place the sequenceout of reading frame and preferably will not create complementaryregions that could produce secondary mRNA structure. Substitutionalvariants are those in which at least one residue has been removed and adifferent residue inserted in its place. Such substitutions generallyare made in accordance with Table 1 and are referred to as conservativesubstitutions.

TABLE 1 Amino Acid Substitutions Original Exemplary ResidueSubstitutions Ala Ser Arg Lys Asn Gln Asp Glu Cys Ser Gln Asn Glu AspGly Pro His Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile PheMet; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions that ingeneral are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl. Modifications in the FcRH can alsoinclude modifications in glycosylation.

In all mutational events, it is understood that the controlling aspectof the mutation is the function that the subsequent protein possesses.The preferred mutations are those that do not detectably change thedesired function or that increase the desired function.

Nucleic Acids

Also provided is an isolated nucleic acid that encodes the FcRH of theinvention. The nucleic acid can be single or double stranded and can beRNA or DNA. More specifically, the invention provides an isolatednucleic acid, comprising a nucleotide sequence that encodes SEQ ID NO:1,SEQ ID NO:21, SEQ ID NO:2. SEQ ID NO: 3, SEQ ID NO:22, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, or SEQ ID NO:6, SEQ ID NO:70, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ IDNO:81, optionally with conservative amino acid substitutions. Optionallythe nucleic acid further encodes a signal sequence (e.g., the signalsequences of SEQ ID NO:29, 30, 31, 32, 71, 75, 79). The isolated nucleicacid optionally encodes the sequences with 80, 85, 90, or 95% identity.More specifically, the invention provides an isolated nucleic acid,comprising a nucleotide sequence of SEQ ID NO:7, SEQ ID NO:13, SEQ IDNO:8, SEQ ID NO:34, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:10, SEQ IDNO:36, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:12, SEQ IDNO:38, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20; SEQ IDNO:40, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ IDNO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ IDNO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:101, or SEQ ID NO:102. Optionally, theisolated nucleic acid can further included bases that encode a signalsequence and thus the nucleotide sequence encoding the extracellularregion or full-length huFcRH1, 2, 3, or 6 can optionally furthercomprise the nucleotide sequence of SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39. Optionally, the isolated nucleic acids for moFcRHsinclude nucleic acid sequences that encode signal sequences as well,including for example, those portions of nucleic acid sequences SEQ IDNO:101, SEQ ID NO:97, SEQ ID NO:94, SEQ ID NO:91, SEQ ID NO:88, SEQ IDNO:84.

Preferably the nucleic acid that encodes the full length FcRH1 includesabout 1290 bases. The nucleic acid that encodes the full length FcRH2includes about 1527 bases, and the nucleic acid that encodes the fulllength FcRH3 includes about 2205 bases.

The invention also provides an isolated nucleic acid comprising asequence that hybridizes under stringent conditions to a hybridizationprobe, wherein the hybridization probe comprises the nucleotide sequenceof SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:8, SEQ ID NO:34, SEQ ID NO:9,SEQ ID NO:14, SEQ ID NO:10, SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:12, SEQ ID NO:38, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, and SEQ ID NO:20; SEQ ID NO:40, SEQ ID NO:84, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ IDNO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ IDNO:101, or SEQ ID NO:102, or the complement of either sequence.

Further provided is a single stranded nucleic acid that hybridizes understringent conditions to a nucleic acid having the sequence of SEQ IDNO:7, SEQ ID NO:13, SEQ ID NO:8, SEQ ID NO:34, SEQ ID NO:9, SEQ IDNO:14, SEQ ID NO:10, SEQ ID NO:36, SEQ ID NO:11, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:12, SEQ ID NO:38, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, and SEQ ID NO:20; SEQ ID NO:40, SEQ ID NO:84, SEQ ID NO:85, SEQID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ IDNO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ IDNO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, or SEQID NO:102.

By “hybridizing under stringent conditions” or “hybridizing under highlystringent conditions” is meant that the hybridizing portion of thehybridizing nucleic acid, typically comprising at least 15 (e.g., 20,25, 30, or 50 nucleotides), hybridizes to all or a portion of theprovided nucleotide sequence under stringent conditions. The term“hybridization” typically means a sequence driven interaction between atleast two nucleic acid molecules, such as a primer or a probe and agene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.Generally, the hybridizing portion of the hybridizing nucleic acid is atleast 80%, for example, at least 90%, 95%, or 98%, identical to thesequence of or a portion of a nucleic acid encoding an FcRH of theinvention, or its complement. Hybridizing nucleic acids of the inventioncan be used, for example, as a cloning probe, a primer (e.g., for PCR),a diagnostic probe, or an antisense probe. Hybridization of theoligonucleotide probe to a nucleic acid sample typically is performedunder stringent conditions. Nucleic acid duplex or hybrid stability isexpressed as the melting temperature or Tm, which is the temperature atwhich a probe dissociates from a target DNA. This melting temperature isused to define the required stringency conditions. If sequences are tobe identified that are related and substantially identical to the probe,rather than identical, then it is useful to first establish the lowesttemperature at which only homologous hybridization occurs with aparticular concentration of salt (e.g., SSC or SSPE). Assuming that a 1%mismatch results in a 1° C. decrease in the Tm, the temperature of thefinal wash in the hybridization reaction is reduced accordingly (forexample, if sequence having >95% identity with the probe are sought, thefinal wash temperature is decreased by 5° C.). In practice, the changein Tm can be between 0.5° C. and 1.5° C. per 1% mismatch. Stringentconditions involve hybridizing at 68° C. in 5×SSC/5×Denhardt'ssolution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature.Moderately stringent conditions include washing in 3×SSC at 42° C. Theparameters of salt concentration and temperature can be varied toachieve the optimal level of identity between the probe and the targetnucleic acid. Additional guidance regarding such conditions is readilyavailable in the art, for example, in Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubelet al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley& Sons, NY) at Unit 2.10.

The nucleic acids of the present invention are optionally labeled,directly or indirectly. Such labeled nucleic acids are useful in variousdiagnostic techniques including for example, in situ hybridization,FISH, in situ PCR, and PRINS. Both methods involve the preparation ofshort sequences of single-stranded nucleic acid probes that arecomplementary to the nucleic acid sequences that encode an FcRH. See,e.g., M Andreeff and D Pinkel (1999), An Introduction to FlourescentIn-Situ Hybridization: Principles and Clinical Applications, John Wiley& Sons, Ltd; Roche Applied Sciences (2000), Nonradioactive In SituHybridization Application Manual; Roche Applied Sciences (1999), PCRManual, 2d edition, which are incorporated in their entirety for methodsof using nucleic acids.

Vectors, Cells, and Methods of Using

Also provided is an expression vector comprising a nucleic acid of theinvention, wherein the nucleic acid is operably linked to an expressioncontrol sequence. A wide variety of expression system/regulatorysequence combinations may be employed in expressing the disclosed. Suchuseful regulatory sequences include, for example, the early or latepromoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system,the trp system, the TAC system, the TRC system, the LTR system, themajor operator and promoter regions of phage lambda, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (for example,Pho5), the AOX 1 promoter of methylotrophic yeast, the promoters of theyeast a-mating factors, and other sequences known to control theexpression of genes of prokaryotic or eukaryotic cells or their viruses,and various combinations thereof.

Such an expression vector can be designed to be expressed by eukaryoticcells or prokaryotic cells. The vectors of the present invention thusprovide DNA molecules which are capable of integration into aprokaryotic or eukaryotic chromosome and expression. The inserted genesin viral and retroviral vectors usually contain promoters, and/orenhancers to help control the expression of the desired gene product. Apromoter is generally a sequence or sequences of DNA that function whenin a relatively fixed location in regard to the transcription startsite. A promoter contains core elements required for basic interactionof RNA polymerase and transcription factors, and may contain upstreamelements and response elements. It has been shown that all specificregulatory elements can be cloned and used to construct expressionvectors that are selectively expressed in specific cell types. Forexample, the glial fibrillary acetic protein (GFAP) promoter has beenused to selectively express genes in cells of glial origin. Expressionvectors used in eukaryotic host cells (e.g., yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

The invention further provides transfer vectors, which include anynucleotide construction used to deliver genes into cells (e.g., aplasmid), or as part of a general strategy to deliver genes, e.g., aspart of recombinant retrovirus or adenovirus (Ram et al. Cancer Res.53:83-88, (1993)). As used herein, plasmid or viral vectors are agentsthat transport the disclosed nucleic acids into the cell withoutdegradation and include a promoter yielding expression of the gene inthe cells into which it is delivered. In some embodiments the FcRHs arederived from either a virus or a retrovirus. Viral vectors include, forexample, Adenovirus, Adeno-associated virus, Herpes virus, Vacciniavirus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis andother RNA viruses, including these viruses with the HIV backbone. Alsopreferred are any viral families that share the properties of theseviruses that make them suitable for use as vectors. Retroviruses includeMurine Maloney Leukemia virus, MMLV, and retroviruses that express thedesirable properties of MMLV as a vector. Retroviral vectors are able tocarry a larger genetic payload, i.e., a transgene or marker gene, thanother viral vectors, and for this reason are a commonly used vector.However, they are not as useful in non-proliferating cells. Adenovirusvectors are relatively stable and easy to work with, have high titers,and can be delivered in aerosol formulation, and can transfectnon-dividing cells. Pox viral vectors are large and have several sitesfor inserting genes, they are thermostable and can be stored at roomtemperature. A preferred embodiment is a viral vector which has beenengineered so as to suppress the immune response of the host organism,elicited by the viral antigens.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines that have been engineered to expressthe gene products of the early genes in trans.

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference. A retrovirus is essentially a packagewhich has packed into it nucleic acid cargo. The nucleic acid cargocarries with it a packaging signal, which ensures that the replicateddaughter molecules will be efficiently packaged within the package coat.In addition to the package signal, there are a number of molecules thatare needed in cis, for the replication, and packaging of the replicatedvirus. Typically a retroviral genome, contains the gag, pol, and envgenes which are involved in the making of the protein coat. It is thegag, pol, and env genes which are typically replaced by the foreign DNAthat it is to be transferred to the target cell. Retrovirus vectorstypically contain a packaging signal for incorporation into the packagecoat, a sequence which signals the start of the gag transcription unit,elements necessary for reverse transcription, including a primer bindingsite to bind the tRNA primer of reverse transcription, terminal repeatsequences that guide the switch of RNA strands during DNA synthesis, apurine rich sequence 5′ to the 3′ LTR that serve as the priming site forthe synthesis of the second strand of DNA synthesis, and specificsequences near the ends of the LTRs that enable the insertion of the DNAstate of the retrovirus to insert into the host genome. The removal ofthe gag, pol, and env genes allows for about 8 kb of foreign sequence tobe inserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line that has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang, Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis, BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virons are generated in a cell line such as thehuman 293 cell line. In another preferred embodiment both the E1 and E3genes are removed from the adenovirus genome.

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorporated by reference for material related tothe AAV vector.

Molecular genetic experiments with large human herpesviruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection withherpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter andRobertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses(herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have thepotential to deliver fragments of human heterologous DNA >150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable The maintenanceof these episomes requires a specific EBV nuclear protein, EBNA1,constitutively expressed during infection with EBV. Additionally, thesevectors can be used for transfection, where large amounts of protein canbe generated transiently in vitro. Herpesvirus amplicon systems are alsobeing used to package pieces of DNA >220 kb and to infect cells that canstably maintain DNA as episomes. Other useful systems include, forexample, replicating and host-restricted non-replicating vaccinia virusvectors.

The invention also provides an isolated cell comprising a vector of theinvention. The isolated cell can be either a eukaryotic or prokaryoticcell, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces;fungi such as yeasts (Saccharomyces, and methylotrophic yeast such asPichia, Candida, Hansenula, and Torulopsis); and animal cells, such asCHO, R1. 1, B-W and LM cells, African Green Monkey kidney cells (forexample, COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (forexample, Sf9), and human cells and plant cells in tissue culture.

Also provided is a method of making a FcRH, or a fragment or variantthereof comprising culturing a cell comprising a vector of the inventionunder conditions permitting expression of the FcRH. The method comprisesculturing a cell comprising an exogeneous nucleic acid that encodes theFcRH, fragment, or variant, wherein the exogeneous nucleic acid isoperably linked to an expression control sequence, and wherein theculture conditions permit expression of the FcRH, fragment, or variantunder the control of the expression control sequence; harvesting themedium from the cultured cells, and isolating the FcRH, fragment, orvariant from the cell or culture medium. Optionally the exogenousnucleic acid is the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:13,SEQ ID NO:8, SEQ ID NO:34, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:10, SEQID NO:36, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:12, SEQ IDNO:38, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; SEQID NO:40, SEQ ID NO: 84, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ IDNO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:101, or SEQ ID NO:102 or a combinationthereof. Optionally, the exogenous nucleic acid further comprises anucleotide sequence that encodes a signal sequence. In the recombinantmethods, the cell can be any known host cell, including for example, aprokaryotic or eukaryotic cell. The nucleic acids that are delivered tocells, generally in a plasmid or other vector, typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product.

Those skilled in the art of molecular biology will understand that awide variety of expression systems may be used to produce recombinantFcRH polypeptides (as well as fragments, fusion proteins, and amino acidsequence variants with therapeutic activity) for use in the methods ofthe invention. Thus, FcRH may be produced using prokaryotic host cells(e.g., Escherichia coli) or eukaryotic host cells (e.g., Saccharomycescerevisiae, insect cells such as Sf9 cells, or mammalian cells such asCHO cells, COS-1, NIH 3T3, or HeLa cells). These cells are commerciallyavailable from, for example, the American Type Culture Collection,Rockville, Md. (see also F. Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y., 1998). The methodof transformation and the choice of expression vector will depend on thehost system selected. Transformation and transfection methods aredescribed, e.g., in Ausubel et al., supra, and expression vectors may bechosen from the numerous examples known in the art.

A nucleic acid sequence encoding an FcRH is introduced into a plasmid orother vector, which is then used to transform living cells. Constructsin which a cDNA containing the entire FcRH coding sequence, a fragmentof the FcRH coding sequence, amino acid variations of the FcRH codingsequence, or fusion proteins of the aforementioned, inserted in thecorrect orientation into an expression plasmid, may be used for proteinexpression. In some cases, for example, it may be desirable to expressthe FcRH coding sequence under the control of an inducible ortissue-specific promoter.

Eukaryotic expression systems permit appropriate post-translationalmodifications to expressed proteins. Thus, eukaryotic, and morepreferably mammalian expression systems, allow glycosylations patternscomparable to naturally expressed FcRH. Transient transfection of aeukaryotic expression plasmid allows the transient production of FcRH bya transfected host cell. FcRH may also be produced by astably-transfected mammalian cell line. A number of vectors suitable forstable transfection of mammalian cells are available to the public(e.g., see Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985,Supp. 1987), as are methods for constructing such cell lines (see e.g.,F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y., 1998). Another preferred eukaryotic expressionsystem is the baculovirus system using, for example, the vectorpBacPAK9, which is available from Clontech (Palo Alto, Calif.). Ifdesired, this system may be used in conjunction with other proteinexpression techniques, for example, the myc tag approach described byEvan et al. (Mol. Cell. Biol. 5:3610-3616, 1985) or analogous taggingapproaches, e.g., using a hemagluttinin (HA) tag.

Once the recombinant protein is expressed, it can be isolated from theexpressing cells by cell lysis followed by protein purificationtechniques such as affinity chromatography. In this example, an antibodythat specifically binds to FcRH, which may be produced by methods thatare well-known in the art, can be attached to a column and used toisolate FcRH. Once isolated, the recombinant protein can, if desired, bepurified further, e.g., by high performance liquid chromatography (HPLC;e.g., see Fisher, Laboratory Techniques In Biochemistry And MolecularBiology, Work and Burdon, Eds., Elsevier, 1980).

Antibodies

The invention also provides a purified antibody or immunologic fragmentthereof, wherein the antibody or fragment thereof selectively binds toan FcRH. As used herein, the term “antibody” encompasses, but is notlimited to, whole immunoglobulin (i.e., an intact antibody) of anyclass. Native antibodies are usually heterotetrameric glycoproteins,composed of two identical light (L) chains and two identical heavy (H)chains. Typically, each light chain is linked to a heavy chain by onecovalent disulfide bond, while the number of disulfide linkages variesbetween the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light andheavy chain variable domains. The light chains of antibodies from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa (k) and lambda (l), based on the amino acid sequences oftheir constant domains. Depending on the amino acid sequence of theconstant domain of their heavy chains, immunoglobulins can be assignedto different classes. There are five major classes of immunoglobulins:IgA, IgD, IgE, IgG and IgM, and several of these may be further dividedinto subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1and IgA-2. The heavy chain constant domains that correspond to thedifferent classes of immunoglobulins are called alpha, delta, epsilon,gamma, and mu, respectively.

The term “variable” is used herein to describe certain portions of thevariable domains that differ in sequence among antibodies and are usedin the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a b-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the b-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies (see Kabat E. A.et al., “Sequences of Proteins of Immunological Interest” NationalInstitutes of Health, Bethesda, Md. (1987)). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “antibody or fragments thereof” can also encompass chimericantibodies and hybrid antibodies, with dual or multiple antigen orepitope specificities, and fragments, such as F(ab′)₂, Fab′, Fab and thelike, including hybrid fragments. Thus, fragments of the antibodies thatretain the ability to bind their specific antigens are provided. Forexample, fragments of antibodies which maintain FcRH binding activityare included within the meaning of the term “antibody or fragmentthereof.” Such antibodies and fragments can be made by techniques knownin the art and can be screened for specificity and activity according tothe methods set forth in the Examples and in general methods forproducing antibodies and screening antibodies for specificity andactivity (See Harlow and Lane. Antibodies, A Laboratory Manual. ColdSpring Harbor Publications, New York, (1988)).

Also included within the meaning of “antibody or fragments thereof” areconjugates of antibody fragments and antigen binding proteins (singlechain antibodies) as described, for example, in U.S. Pat. No. 4,704,692,the contents of which are hereby incorporated by reference.

In one embodiment, the antibody is a monoclonal antibody. The term“monoclonal antibody” as used herein refers to an antibody obtained froma substantially homogeneous population of antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired activity (See, U.S. Pat. No. 4,816,567 and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

Monoclonal antibodies of the invention may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) or Harlow and Lane, Antibodies, A Laboratory Manual. Cold SpringHarbor Publications, New York, (1988). In a hybridoma method, a mouse orother appropriate host animal, is typically immunized with an immunizingagent to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro. Preferably,the immunizing agent comprises an FcRH. Traditionally, the generation ofmonoclonal antibodies has depended on the availability of purifiedprotein or peptides for use as the immunogen. More recently DNA basedimmunizations have shown promise as a way to elicit strong immuneresponses and generate monoclonal antibodies. In this approach,DNA-based immunization can be used, wherein DNA encoding a portion ofFcRH, preferably the N- or C-terminal region, is injected into the hostanimal according to methods known in the art.

Generally, either peripheral blood lymphocytes (“PBLs”) are used inmethods of producing monoclonal antibodies if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, “MonoclonalAntibodies: Principles and Practice” Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,including myeloma cells of rodent, bovine, equine, and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., “Monoclonal Antibody Production Techniques and Applications” MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against anFcRH. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art, and are described further in Harlow and Lane“Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, NewYork, (1988).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution or FACS sorting procedures and grown bystandard methods. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, plasmacytoma cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also may be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murine sequences(U.S. Pat. No. 4,816,567) or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody comprising one antigen-combining site havingspecificity for FcRH and another antigen-combining site havingspecificity for a different antigen.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994,U.S. Pat. No. 4,342,566, and Harlow and Lane, Antibodies, A LaboratoryManual, Cold Spring Harbor Publications, New York, (1988). Papaindigestion of antibodies typically produces two identical antigen bindingfragments, called Fab fragments, each with a single antigen bindingsite, and a residual Fc fragment. Pepsin treatment yields a fragment,called the F(ab′)₂ fragment, that has two antigen combining sites and isstill capable of cross-linking antigen.

The Fab fragments produced in the antibody digestion also contain theconstant domains of the light chain and the first constant domain of theheavy chain. Fab′ fragments differ from Fab fragments by the addition ofa few residues at the carboxy terminus of the heavy chain domainincluding one or more cysteines from the antibody hinge region. TheF(ab′)₂ fragment is a bivalent fragment comprising two Fab′ fragmentslinked by a disulfide bridge at the hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. Antibody fragments originallywere produced as pairs of Fab′ fragments which have hinge cysteinesbetween them. Other chemical couplings of antibody fragments are alsoknown.

An isolated immunogenically specific epitope or fragment of the antibodyis also provided. A specific immunogenic epitope of the antibody can beisolated from the whole antibody by chemical or mechanical disruption ofthe molecule. The purified fragments thus obtained can be tested todetermine their immunogenicity and specificity by the methods taughtherein. Immunoreactive epitopes of the antibody can also be synthesizeddirectly. An immunoreactive fragment is defined as an amino acidsequence of at least about two to five consecutive amino acids derivedfrom the antibody amino acid sequence.

One method of producing proteins comprising the antibodies of thepresent invention is to link two or more peptides or polypeptidestogether by protein chemistry techniques. For example, peptides orpolypeptides can be chemically synthesized using currently availablelaboratory equipment using either Fmoc (9-fluorenylmethyl-oxycarbonyl)or Boc (tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc.,Foster City, Calif.). One skilled in the art can readily appreciate thata peptide or polypeptide corresponding to the antibody of the presentinvention, for example, can be synthesized by standard chemicalreactions. For example, a peptide or polypeptide can be synthesized andnot cleaved from its synthesis resin whereas the other fragment of anantibody can be synthesized and subsequently cleaved from the resin,thereby exposing a terminal group that is functionally blocked on theother fragment. By peptide condensation reactions, these two fragmentscan be covalently joined via a peptide bond at their carboxyl and aminotermini, respectively, to form an antibody, or fragment thereof. (GrantG A (1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y.(1992); Bodansky M and Trost B., Ed. (1993) Principles of PeptideSynthesis. Springer-Verlag Inc., NY). Alternatively, the peptide orpolypeptide can by independently synthesized in vivo as described above.Once isolated, these independent peptides or polypeptides may be linkedto form an antibody or fragment thereof via similar peptide condensationreactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentscan allow relatively short peptide fragments to be joined to producelarger peptide fragments, polypeptides or whole protein domains(Abrahmsen L et al., Biochemistry, 30:4151 (1991)). Alternatively,native chemical ligation of synthetic peptides can be utilized tosynthetically construct large peptides or polypeptides from shorterpeptide fragments. This method consists of a two step chemical reaction(Dawson et al. Synthesis of Proteins by Native Chemical Ligation.Science, 266:776-779 (1994)). The first step is the chemoselectivereaction of an unprotected synthetic peptide-α-thioester with anotherunprotected peptide segment containing an amino-terminal Cys residue togive a thioester-linked intermediate as the initial covalent product.Without a change in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site. Application of this native chemical ligationmethod to the total synthesis of a protein molecule is illustrated bythe preparation of human interleukin 8 (IL-8) (Baggiolini M et al.(1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J. Biol. Chem.,269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991);Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).

Alternatively, unprotected peptide segments can be chemically linkedwhere the bond formed between the peptide segments as a result of thechemical ligation is an unnatural (non-peptide) bond (Schnolzer, M etal. Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

The invention also provides fragments of antibodies that havebioactivity. The polypeptide fragments of the present invention can berecombinant proteins obtained by cloning nucleic acids encoding thepolypeptide in an expression system capable of producing the polypeptidefragments thereof, such as an adenovirus or baculovirus expressionsystem. For example, one can determine the active domain of an antibodyfrom a specific hybridoma that can cause a biological effect associatedwith the interaction of the antibody with FcRH. For example, amino acidsfound to not contribute to either the activity or the bindingspecificity or affinity of the antibody can be deleted without a loss inthe respective activity.

For example, amino or carboxy-terminal amino acids can be sequentiallyremoved from either the native or the modified non-immunoglobulinmolecule or the immunoglobulin molecule and the respective activityassayed in one of many available assays. In another example, a fragmentof an antibody can comprise a modified antibody wherein at least oneamino acid has been substituted for the naturally occurring amino acidat a specific position, and a portion of either amino terminal orcarboxy terminal amino acids, or even an internal region of theantibody, has been replaced with a polypeptide fragment or other moiety,such as biotin, which can facilitate in the purification of the modifiedantibody. For example, a modified antibody can be fused to a maltosebinding protein, through either peptide chemistry of cloning therespective nucleic acids encoding the two polypeptide fragments into anexpression vector such that the expression of the coding region resultsin a hybrid polypeptide. The hybrid polypeptide can be affinity purifiedby passing it over an amylose affinity column, and the modified antibodyreceptor can then be separated from the maltose binding region bycleaving the hybrid polypeptide with the specific protease factor Xa.(See, for example, New England Biolabs Product Catalog, 1996, pg. 164).Similar purification procedures are available for isolating hybridproteins from eukaryotic cells as well.

The fragments, whether attached to other sequences, can also includeinsertions, deletions, substitutions, or other selected modifications ofparticular regions or specific amino acids residues, provided theactivity of the fragment is not significantly altered or impairedcompared to the nonmodified antibody or antibody fragment. Thesemodifications can provide for some additional property, such as toremove or add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antigen. (Zoller M J et al.Nucl. Acids Res. 10:6487-500 (1982).

As used herein, the phrase “specific binding” or “selective binding”refers to a binding reaction which is determinative of the presence ofthe FcRH in a heterogeneous population of proteins and other biologics.Thus, under designated conditions, the antibodies or fragments thereofof the present invention bind to a particular FcRH (e.g., human FcRH 1or any variant thereof), fragment, or variant thereof and do not bind ina significant amount to other proteins (e.g., human FcRH 2, 3, 4, 5, or6), present in the subject. The absence of binding in the presentinvention is considered to be binding that is less than 1.5 timesbackground (i.e., the level of non-specific binding or slightly abovenon-specific binding levels),

Selective binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein,variant, or fragment. In one embodiment the purified antibodyselectively binds to the FcRH comprising a cytoplasmic region with morethan 107 or less than 104 amino acids, a transmembrane region, and anextracellular region. More specifically, the antibody in alternativeembodiments selectively binds FcRH1 but not FcRH2-6; selectively bindsFcRH2 but not 1 or 3-6; selectively binds FcRH3 but not FcRH1-2 or 4-6;selectively binds FcRH6 but not 1-5. Thus, as one embodiment, theantibody selectively binds a polypeptide comprising the amino acidsequence of SEQ ID NO:1, 21, or 2, or a subset thereof, but not topolypeptides comprising the amino acid of SEQ ID NO:3, 22, 4, 5, 23, 24,6, 25, 26, 27, 28, or a subset thereof. In another embodiment thepurified antibody binds to the FcRH comprising the amino acid sequenceof SEQ ID NO:3, 22, or 4, but not to the FcRH comprising the amino acidof SEQ ID NO:1, 21, 2, 5, 23, 24, 6, 25, 26, 27, or 28. In yet anotherembodiment, the purified antibody that binds to the FcRH comprising theamino acid sequence of SEQ ID NO:5, 23, 24, or 6, but not to the FcRHcomprising the amino acid of SEQ ID NO:1, 21, 2, 3, 22, 4, 26, 27, 28.Similarly, the antibodies of the present invention may bind onlymoFcRH1, but not moFcRH 2 or moFcRH3; may bind only FcRH2 and not FcRH1or FcRH3, and may bind only FcRH3 and not FcRH1 or FcRH2.

In certain embodiments, the antibody binds the extracellular region ofone or more FcRHs and in other embodiments the antibody binds thecytoplasmic region of one or more FcRHs. In other embodiments theantibody may selectively bind one isoform of a FcRH. For example, theantibody may bind a polypeptide having the amino acid sequence of SEQ IDNO:23 but not the SEQ ID NO:24 or vice versa. Furthermore, the antibodycan bind to moFcRH1 having the amino acid sequence of SEQ ID NO:70, butnot to a moFcRH1 having amino acid sequence of SEQ ID NO:68. Theantibody may selectively bind a moFcRH2 with a transmembrane region(e.g., having amino acid sequence of SEQ ID NO:73), but not bind to amoFcRH2 lacking a transmembrane region (e.g., having the amino acidsequence of 77). Optionally the antibody of the invention canselectively bind moFcRH but not human, or vice versa.

A variety of immunoassay formats may be used to select antibodies thatselectively bind with a particular protein, variant, or fragment. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a protein, variant, orfragment thereof. See Harlow and Lane. Antibodies, A Laboratory Manual.Cold Spring Harbor Publications, New York, (1988), for a description ofimmunoassay formats and conditions that could be used to determineselective binding. The binding affinity of a monoclonal antibody can,for example, be determined by the Scatchard analysis of Munson et al.,Anal. Biochem., 107:220 (1980).

The invention also provides an antibody reagent kit comprising theantibody or fragment thereof of the invention and reagents for detectingbinding of the antibody or fragment thereof to a ligand. The kit canfurther comprise containers containing the antibody or fragment thereofof the invention and containers containing the reagents. Preferably theligand is a FcRH, variant, or fragment thereof. Particularly, the kitcan detect the presence of one or more FcRHs specifically reactive withthe antibody or an immunoreactive fragment thereof. The kit can includean antibody bound to a substrate, a secondary antibody reactive with theantigen and a reagent for detecting a reaction of the secondary antibodywith the antigen. Such a kit can be an ELISA kit and can comprise thesubstrate, primary and secondary antibodies when appropriate, and anyother necessary reagents such as detectable moieties, enzyme substratesand color reagents as described above. The diagnostic kit can,alternatively, be an immunoblot kit generally comprising the componentsand reagents described herein. Alternatively, the kit could be aradioimmunoassay kit, a Western blot assay kit, an immunohistologicalassay kit, an immunocytochemical assay kit, a dot blot assay kit, afluorescence polarization assay kit, a scintillation proximity assaykit, a homogeneous time resolved fluorescence assay kit, or a BIACORE®analysis kit (Pharmacia, Sweden).

As used throughout, methods of detecting an FcRH or antigen/antibodycomplexes, including complexes comprising an FcRH and optionally theantibody of the present invention, can comprise an ELISA (competition orsandwich), a radioimmunoassay, a Western blot assay, animmunohistological assay, an immunocytochemical assay, a dot blot assay,a fluorescence polarization assay (Jolley (1981); Jiskoot et al (1991);Seethala et al. (1998); Bicamumpaka et al. (1998)), a scintillationproximity assay (Amersham Life Science (1995) Proximity News. Issue 17;Amersham Life Science (1995) Proximity News. Issue 18; Park et al.(1999)), a homogeneous time-resolved fluorescence assay (Park et al.(1999); Stenroos et al. (1988); Morrison, 1988)), or a BIACORE®(Pharmacia, Sweden) analysis Fägerstam et al. (1992) Chromatography597:397-410. Preferably, the antigen/antibody complex is detectablytagged either directly or indirectly. Any desired tag can be utilized,such as a fluorescent tag, a radiolabel, a magnetic tag, or an enzymaticreaction product.

Optionally, the antibody or fragment is a humanized antibody or a fullyhuman antibody. For example, the antibodies can also be generated inother species and “humanized” for administration to humans.Alternatively, fully human antibodies can also be made by immunizing amice or other species capable of making a fully human antibody (e.g.,mice genetically modified to produce human antibodies), screening clonesthat bind FcRH. See, e.g., Lonberg and Huszar (1995) Human antibodiesfrom transgenic mice, Int. Rev. Immunol. 13:65-93, which is incorporatedherein by reference in its entirety for methods of producing fully humanantibodies. As used herein, the term “humanized” and “fully human” inrelation to antibodies, relate to any antibody which is expected toelicit a therapeutically tolerable weak immunogenic response in a humansubject.

Humanized forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′ F(ab′)₂, or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residues thatare found neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanized antibody will comprisesubstantially all or at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important in order to reduceantigenicity. According to the “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework (FR) for the humanized antibody (Sims et al., J. Immunol.,151:2296 (1993) and Chothia et al., J. Mol. Biol., 196:901 (1987)).Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding(see, WO 94/04679 published 3 Mar. 1994).

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region (J(H)) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggemann et al., Year in Immuno., 7:33 (1993)). Humanantibodies can also be produced in phage display libraries (Hoogenboomet al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). The techniques of Cote et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., J. Immunol., 147(1):86-95 (1991)).

In one embodiment, the antibody or fragment thereof is a single chainantibody. In another embodiment, the antibody or fragment is labeled.Optionally the antibody or fragment is conjugated or fused with a toxinor fragment thereof. Examples of the toxin or toxin moiety includediphtheria, ricin, and modifications thereof.

Diagnosis and Treatment

The invention provides uses of the reagents described herein in in vitroand in vivo methods of diagnosing and treating a malignancy ofhematopoietic cell lineage or an autoimmune disease in a subject. Thereagents of the present invention are also useful in screening fordisease manifestations. Such screening may be useful even before theonset of other clinical symptoms and could be used to screening subjectsat risk for disease, so that prophylactic treatment can be startedbefore the manifestation of other signs or symptoms.

By “malignancy” is meant a tumor or neoplasm whose cells possess one ormore nuclear or cytoplasmic abnormalities, including, for example, highnuclear to cytoplasmic ratio, prominent nucleolar/nucleoli variations,variations in nuclear size, abnormal mitotic figures, ormultinucleation. “Malignancies of hematopoietic cell lineage” include,but are not limited to, myelomas, leukemias, lymphomas (Hodgkin's andnon-Hodgkin's forms), T-cell malignancies, B-cell malignancies, andlymphosarcomas or other malignancies described in the REALclassification system or the World Health Organization Classification ofHematologic Malignancies. It should be noted that the absence orpresence of specific FcRHs can be diagnostic for a particular malignancyof hematopoietic cell linage or can be diagnostic for a particular formof a malignancy (e.g., a specific form of leukemia).

By “inflammatory and autoimmune diseases” illustratively includingsystemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis,graft-versus-host disease, Sjögren's syndrome, pernicious anemia,Addison disease, scleroderma, Goodpasture's syndrome, Crohn's disease,autoimmune hemolytic anemia, sterility, myasthenia gravis, multiplesclerosis, Basedow's disease, thrombopenia purpura, insulin-dependentdiabetes mellitus, allergy; asthma, atopic disease; arteriosclerosis;myocarditis; cardiomyopathy; glomerular nephritis; hypoplastic anemia;rejection after organ transplantation and numerous malignancies of lung,prostate, liver, ovary, colon, cervix, lymphatic and breast tissues.

Specifically, the diagnostic methods comprise the steps of contacting abiological sample of the subject with an antibody or nucleic acid of theinvention under conditions that allow the antibody to bind to cells ofhematopoietic cell lineage or allow the nucleic acid to hybridize,preferably under stringent conditions, with nucleic acids of thebiological sample; and detecting the amount or pattern of binding.Changes in the amount or pattern of binding as compared to binding in acontrol sample indicate a malignancy or an inflammatory or autoimmunedisease.

In various embodiments, the antibody used in the diagnostic method canselectively bind with an FcRH having the amino acid sequence of SEQ IDNO:1, 21, 2, 3, 22, 4, 5, 24, or 6.

The detecting step of the diagnostic method can be selected from methodsroutine in the art. For example, the detection step can be performed invivo using a noninvasive medical technique such as radiography,fluoroscopy, sonography, imaging techniques such as magnetic resonanceimaging, and the like. In vitro detection methods can be used to detectbound antibody or fragment thereof in an ELISA, RIA,immunohistochemically, FACS, IHC, FISH, or similar assays.

As used throughout, “biological sample” refers to a sample from anyorganism. The sample can be, but is not limited to, peripheral blood,plasma, urine, saliva, gastric secretion, feces, bone marrow specimens,primary tumors, embedded tissue sections, frozen tissue sections, cellpreparations, cytological preparations, exfoliate samples (e.g.,sputum), fine needle aspirations, amnion cells, fresh tissue, drytissue, and cultured cells or tissue. It is further contemplated thatthe biological sample of this invention can also be whole cells or cellorganelles (e.g., nuclei). The sample can be unfixed or fixed accordingto standard protocols widely available in the art and can also beembedded in a suitable medium for preparation of the sample. Forexample, the sample can be embedded in paraffin or other suitable medium(e.g., epoxy or acrylamide) to facilitate preparation of the biologicalspecimen for the detection methods of this invention.

The invention also provides a method of treating a malignancy ofhematopoietic cell lineage or an inflammatory or autoimmune disease in asubject, comprising contacting the subject's malignant cells orinflammatory cells with a therapeutically effective amount of a reagent(e.g., an antibody or nucleic acid) or a therapeutic composition of areagent of the invention. The contacting step can occur byadministration of the reagent or composition using any number of meansavailable in the art. Typically, the reagent or composition isadministered to the subject transdermally (e.g., by a transdermal patchor a topically applied cream, ointment, or the like), orally,subcutaneously, intrapulmonaryily, transmucosally, intraperitoneally,intrauterinely, sublingually, intrathecally, intramuscularly,intraarticularly, etc. using conventional methods. In addition, thereagent or composition can be administered via injectable depot routessuch as by using 1-, 3-, or 6-month depot injectable or biodegradablematerials and methods.

Regardless of the route of administration, the amount of the reagentadministered or the schedule for administration will vary amongindividuals based on age, size, weight, condition to be treated, mode ofadministration, and the severity of the condition. One skilled in theart will realize that dosages are best optimized by the practicingphysician and methods for determining dosage are described, for examplein Remington's Pharmaceutical Science, latest edition. Guidance inselecting appropriate doses for antibodies is found in the literature ontherapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies,Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch.22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis andTherapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. Atypical dose of the antibody used alone might range from about 1 μg/kgto up to 100 mg/kg of body weight or more per day, and preferably 1μg/kg to up to 1 mg/kg, depending on the factors mentioned above. Anintravenous injection of the antibody or fragment thereof, for example,could be 10 ng-1 g of antibody or fragment thereof, and preferably 10ng-1 mg depending on the factors mentioned above. For local injection, atypical quantity of antibody ranges from 1 pg to 1 mg. Preferably, thelocal injection would be at an antibody concentration of 1-100 μg/ml,and preferably 1-20 μg/ml.

The nucleic acids of the invention can delivered to cells in a varietyof ways. For example, if the nucleic acid of this invention is deliveredto the cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10⁷ to 10⁹plaque forming units (pfu) per injection, but can be as high as 10¹² pfuper injection. Ideally, a subject will receive a single injection. Ifadditional injections are necessary, they can be repeated at six monthintervals for an indefinite period and/or until the efficacy of thetreatment has been established. As set forth herein, the efficacy oftreatment can be determined by evaluating the clinical parameters.

The exact amount of the nucleic acid or vector required will vary asdescribed above. Thus, it is not possible to specify an exact amount forevery nucleic acid or vector. An appropriate amount can be determined byone of ordinary skill in the art using only routine experimentationgiven the teachings herein.

The invention further provides a therapeutic composition of the reagentof the invention. Such a composition typically contains from about 0.1to 90% by weight (such as 1 to 20% or 1 to 10%) of a therapeutic agentof the invention in a pharmaceutically acceptable carrier. Solidformulations of the compositions for oral administration may containsuitable carriers or excipients, such as corn starch, gelatin, lactose,acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalciumphosphate, calcium carbonate, sodium chloride, or alginic acid.Disintegrators that can be used include, without limitation,microcrystalline cellulose, corn starch, sodium starch, glycolate, andalginic acid. Tablet binders that may be used include acacia,methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolindone(Povidone), hydroxypropyl methylcellulose, sucrose, starch, andethylcellulose. Lubricants that may be used include magnesium stearates,stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica.

Liquid formulations for oral administration prepared in water or otheraqueous vehicles may contain various suspending agents such asmethylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan,acacia, polyvinylpyrrolidone, and polyvinyl alcohol. The liquidformulations may also include solutions, emulsions, syrups and elixirscontaining, together with the active compound(s), wetting agents,sweeteners, and coloring and flavoring agents. Various liquid and powderformulations can be prepared by conventional methods for inhalation intothe lungs of the mammal to be treated.

Injectable formulations of the compositions may contain various carrierssuch as vegetable oils, dimethylacetamide, dimethylformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, liquid polyethylene glycol, and the like).For intravenous injections, water soluble version of the compounds maybe administered by the drip method, whereby a pharmaceutical formulationcontaining the antifungal agent and a physiologically acceptableexcipient is infused. Physiologically acceptable excipients may include,for example, 5% dextrose, 0.9% saline, Ringer's solution or othersuitable excipients. Intramuscular preparations, e.g., a sterileformulation of a suitable soluble salt form of the compounds, can bedissolved and administered in a pharmaceutical excipient such aswater-for-injection, 0.9% saline, or 5% glucose solution. A suitableinsoluble form of the compound may be prepared and administered as asuspension in an aqueous base or a pharmaceutically acceptable oil base,such as an ester of a long chain fatty acid (e.g., ethy; oleate).

A topical semi-solid ointment formulation typically contains aconcentration of the active ingredient from about 1 to 20%, e.g., 5 to10%, in a carrier such as a pharmaceutical cream base. Variousformulations for topical use include drops, tinctures, lotions, creams,solutions, and ointments containing the active ingredient and varioussupports and vehicles. The optimal percentage of the therapeutic agentin each pharmaceutical formulation varies according to the formulationitself and the therapeutic effect desired in the specific pathologiesand correlated therapeutic regimens.

The effectiveness of the method of treatment can be assessed bymonitoring the patient for known signs or symptoms of the conditionsbeing treated. For example, in the treatment of a malignancy ofhematopoietic cell lineage, the reduction or stabilization of the numberof abnormally proliferative cells would indicate successful treatment.In the treatment of arthritis, for example, a reduction in the amount ofjoint inflammation would indicate successful treatment. Thus, by“therapeutically effective” is meant an amount that provides the desiredtreatment effect.

The invention further provides a method of modulating a humoral immuneresponse in a subject, comprising administering to the subject anisolated FcRH, an antibody, or nucleic acid of the invention. By“modulation” is meant either up-regulating or down-regulating. Thus, inthe case of an allergic response, one skilled in the art would choose todown-regulate the humoral immune response. In the case of exposure of asubject to an infectious agent (e.g., a viral or bacterial agent), oneskilled in the art would choose to upregulate the humoral antibodyresponse.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1 Identification of FcRH1, FcRH2, and FcRH3

In order to isolation of FcRH cDNA Clones, rapid amplification of cDNAends (RACE)-PCR was performed by using a MARATHON-READY human lymph nodecDNA library (CLONTECH, Mountain View, Calif.). Gene-specific primerswere as follows: FcRH3, forward 5′-TGAGTCTCAGGGTCACAGTTCCG-3′ (SEQ IDNO:41) and reverse 5′-GCTCTTGAACTTGGATATTTAGGGGT-3′ (SEQ ID NO:42);FcRH2, forward 5′-CCAGTGTATGTCAATGTGGGCTCTG-3′ (SEQ ID NO:43) andreverse 5′-CGTTGAAAGAGCTCTTGGACTTTTATC-3′ (SEQ ID NO:44); and FcRH1,forward 5′-GCCTCAAAAGAAAAATAGGAAGACGTT-3′ (SEQ ID NO:45) and reverse5′-AAGCTCACATCAGCGACAGGGAC-3′ (SEQ ID NO:46). RACE products weresubjected to a second round of nested PCR and visualized by agarose gelelectrophoresis and ethidium bromide staining.

Primers used in end-to-end amplification to generate full-length cDNAswere as follows: FcRH3, forward 5′-TCTTGGAGATAAGTCGGGCTTT-3′ (SEQ IDNO:47) and reverse 5′-ATCCTGCAGCCCAGCCTCGTAGGAG-3′ (SEQ ID NO:48);FcRH2, forward 5′-GGTCCTCATGCTGCTGTGGTCATT-3′ (SEQ ID NO:49) and reverse5′-GCTGTTGATCTTCCCTTCTGATTC-3′ (SEQ ID NO:50); and FcRH1, forward5′-ATGCTGCCGAGGCTGTTGCTGTTG3′ (SEQ ID NO:51) and reverse5′-CATAGCATCTTCATAGTCCACATC-3′ (SEQ ID NO:52). Each amplificationreaction underwent initial denaturation of 94° C. for 30 s followed by30 cycles of denaturation at 94° C. for 5 s and annealing at 68° C. for4 min, and final extension at 72° C. for 6 min.

PCR products were ligated into the pCR2.1 TOPO T/A vector (Invitrogen,Carlsbad, Calif.). Inserts were DNA-sequenced on both strands by thedideoxy chain termination method using Thermo Sequenase (AmershamPharmacia, Piscataway, N.J.) and an automated sequencer (Li-Cor,Lincoln, Nebr.). Nucleotide and amino acid sequence alignment wasanalyzed with a DNASTAR™ (DNAStar, Madison, Wis.) software package, andhomology searches were performed by using BLAST (Altschul, S. F. et al.(1990) J. Mol. Biol. 215, 403-410).

RNA blot analysis was subsequently performed. Northern blots (CLONTECH)were hybridized with 32P-dCTP-labeled probes: a 528-bp EcoRI fragmentcorresponding to the 5′ untranslated (UT)-EC1 regions of the FcRH3 cDNA,a 200-bp PCR product corresponding to a portion of the 3′ UT region ofthe FcRH2 cDNA, and a 257-bp PCR product corresponding to a portion ofthe 3′ UT region of the FcRH1 cDNA. Membranes were hybridized for 1 h at65° C., washed, and exposed to x-ray film (Kubagawa, H. et al. (1997)Proc. Natl. Acad. Sci. USA 94, 5261-5266).

Reverse transcription (RT)-PCR was performed. Human tonsillar cells,obtained with Institutional Review Board approval, were separated intoCD19+ and CD19 subpopulations by magnetic cell sorting (Miltenyi Biotec,Auburn, Calif.). Viable CD19+ cells were stained with FITC-labeledanti-CD38 (Immunotech, Westbrook, Me.) and phycoerythrin-labeledanti-IgD mAbs (Southern Biotechnology Associates, Birmingham, Ala.)before sorting cells with a FACSTARPLUS™ instrument (Becton Dickinson,Franklin Lakes, N.J.) into TRIZOL® reagent (Life Technologies, GrandIsland, N.Y.) for RNA isolation. Total cellular RNA was primed withrandom hexamers and oligo(dT) primers and reverse-transcribed withSUPERSCRIPT™ II (Invitrogen, Carlsbad, Calif.) into single-strandedcDNA. RT-PCR was performed by using RNA from tonsillar B cells and celllines, with GIBCO/BRL Taq polymerase (Life Technologies). The followinggene-specific primer pairs were used in the RT-PCR analysis of FcRH1-5expression in cell lines and tonsillar B cell subpopulations: FcRH1forward, 5′-CTC AAC TTC ACA GTG CCT ACT GGG-3′ (SEQ ID NO:53) andreverse, 5′-TCC TGC AGA GTC ACT AAC CTT GAG-3′ (SEQ ID NO:54); FcRH2forward, 5′-CCA GTG TAT GTC AAT GTG GGC TCT G (SEQ ID NO:55) andreverse, 5′-CAT TCT TCC CTC AAA TCT TTA CAC-3′ (SEQ ID NO:56); FcRH3forward, 5′-CAG CAC GTG GAT TCG AGT CAC-3′ (SEQ ID NO:57) and reverse,5′-CAG ATC TGG GAA TAA ATC GGG TTG-3′ (SEQ ID NO:58) FcRH4 forward,5′-TCT TCA GAG ATG GCG AGG TCA-3′ (SEQ ID NO:59) and reverse, 5′-TTT TGGGGT GTA CAT CAA CAT ACA AG-3′ (SEQ ID NO:60); and FcRH forward, 5′-TGTTGC CCT GTT TCT TCC AAT ACA-3′ (SEQ ID NO:61) and reverse, 5′-CAG AGTTGG CCG ACC TAC GC-3′ (SEQ ID NO:62). Each amplification reactionunderwent initial denaturation at 94° for 5 min followed by 35 cycles ofdenaturation at 94° for 30 s, annealing at 60° for 30 s, extension at72° for 1 min, and final extension at 72° for 7 min. Amplified productswere visualized in 1% agarose gels containing ethidium bromide anddocumented with the BIO-RAD FLUOR-S™ Imager (Bio-Rad Laboratories,Hercules, Calif.).

The following human cell lines were used: REH and Nalm 16 pro-B celllines (Korsmeyer, S. J. et al. (1983) J. Clin. Invest. 71, 301-313);697, 207, and OB5 pre-B cell lines (Findley, H. W. et al. (1982) Blood60, 1305-1309; Martin, D. et al. (1991) J. Exp. Med. 173, 639-645);Ramos, Daudi, and Raji B cell lines (Pulvertaft, R. J. V. (1964) Lancet1, 238-240; Klein, E. et al. (1968) Cancer Res. 28, 1300-1310; Klein, G.et al. (1975) Intervirology 5, 319-33431-33); THP-1 and U937 monocytoidcell lines, HL-60 promyelocytic and KG-1 myelocytic cell lines, Jurkat Tcell line and the K₅₆₂ erythroid cell line (American Type CultureCollection).

A consensus sequence was generated that corresponds to theGenBank-derived amino terminal sequences of the second Ig-like domainsof FcR (FcγRI and FcγRII/III) and the third Ig-like domain of thepolymeric Ig receptor:GEPIXLRCHSWKDKXLXKVTYXQNGKAXKFFH (SEQ ID NO:63). Asearch of the National Center for Biotechnology Information proteindatabase with this sequence identified two overlapping human genomicbacterial artificial chromosome (BAC) clones, AL135929 and AL356276,which are located at 1q21.2-22. The second clone contained threeputative Ig superfamily genes encoding complementary amino acidsequences that were designated FcRH, FcRH2, and FcRH3. See FIG. 1. Thepredicted amino acid sequences of these gene segments shared 23-57%identity with each other and 14-28% identity with human FcγRI (CD64).Further analysis of the FcRH locus led to the identification of twoadditional genes (FcRH4, and FcRH5) and one pseudogene (FcRH4ψ),immediately centromeric of FcRH1-3, two of which have recently beendescribed as IRTA1 (FcRH4) and IRTA2 (FcRH5) (Hatzivassiliou, G. et al.(2001) Immunity 14, 277-289).

To determine whether these genes are expressed by lymphocytes, thepredicted amino acid sequences of their protein products were used tosearch the Lymphochip expressed sequence tag database with the TBLASTNalgorithm (Alizadeh, A. A. et al. (2000) Nature (London) 403, 503-511).Two expressed sequence tags (AA505046 and AA282433) were identified thatshare complete identity over 23 amino acids in their translated ORFswith the N terminus of FcRH1. Lymphochip microarray data analysisindicated that these expressed sequence tags are expressed at relativelyhigh levels in peripheral lymphoid tissues, including the lymph nodes,tonsils, resting peripheral B cells, and normal germinal center (GC) Bcells. Among the different lymphoid malignancies, their expressionproved to be highest in chronic lymphocytic leukemias, follicularlymphomas, and some diffuse large cell lymphomas of B lineage.

FcRH1, FcRH2, and FcRH3 cDNAs were isolated by RACE-PCR from a humanlymph node cDNA library in both 5′ and 3′ directions. Full-length cDNAsof the coding regions for FcRH1, FcRH2, and FcRH3 were obtained byend-to-end PCR using unique primers generated from the cDNA sequencesdelineated for the 5′ UT and 3′UT regions. Southern blot analysis ofhuman genomic DNA digested with BamHI, EcoRI, or HindIII using cDNAprobes specific for the 3′ UT regions of each cDNA revealed either oneor two hybridizing fragments, suggesting that FcRH1, FcRH2, and FcRH3are encoded by single genes. Analysis of full-length cDNA sequencesindicated that FcRH1, FcRH2, and FcRH3 have ORFs of 1,287 bp, 1,524 bp,and 2,202 bp, respectively, and encode type I transmembrane proteins of429 aa, 508 aa, and 734 aa, respectively. Based on predicted consensussignal peptide cleavage sites (Von Heijne, G. (1986) Nucleic Acid Res.14, 4683-4690; Nielsen, H. (1997) Protein Eng. 10, 1-6), the relativecore peptide molecular masses were estimated as 45,158 for FcRH1, 53,407for FcRH2, and 78,849 for FcRH3. These type I transmembrane proteinspossess 3-6 extracellular C2 (Williams, A. F. & Barclay, A. N. (1988)Annu. Rev. Immunol. 6, 381-405; Bork, P. et al. (1994) J. Mol. Biol.242, 309-320; Vaughn, D. E. & Bjorkman, P. J. (1996) Neuron 16, 261-273)type Ig-like domains with 3-7 potential N-linked glycosylation sites,uncharged transmembrane segments, and relatively long cytoplasmic tailscontaining consensus motifs for ITIMs and/or ITAMs. See FIG. 2A.

Multiple alignment analysis of the translated cDNAs, using FcRH3 as theindex sequence of comparison, indicates that FcRH1, FcRH2, and FcRH3have highly conserved hydrophobic signal peptides and correspondingIg-like extracellular domains (FIG. 2B). Their hydrophobic transmembrane(uncharged with the exception of FcRH1 which includes an acidic domain)domains (Sonnhammer, E. L. L. et al. (1998) in A Hidden Markov Model forPredicting Transmembrane Helices in Protein Sequences, eds. Glasgow, J.,Littlejohn, T., Major, F., Lathrop, R., Sankoff, D. & Sensen, C. (Am.Assoc. for Artificial Intelligence, Menlo Park, Calif.), pp. 175-182)are also well conserved, but their cytoplasmic domains are not. FcRH1has a long cytoplasmic tail containing three potential ITAMs, the firstand third of which fit the consensus sequence(E/D)-X-X-Y-X-X-(L/I)-X₆₋₈-Y-X-X-(L/I) (SEQ ID NO:64, with six aminoacid between the consensus sequences; SEQ ID NO:65, with seven aminoacid residues between the consensus sequences; and SEQ ID NO:66, witheight amino acid residues between the consensus sequences), whereas, thesecond has only one tyrosine residue. The shorter cytoplasmic domain ofFcRH2 contains one potential ITAM and two ITIM consensus sequences(I/V/L/S)-X-Y-X-X-(L/V) (SEQ ID NO:67) separated by 22 amino acids.FcRH3 has the longest cytoplasmic tail. It contains one potential ITAM,one ITIM, and another potential ITAM that also has a single tyrosineresidue.

An RNA blot analysis with gene-specific probes was performed on 16 humantissues, including six primary or secondary lymphoid tissues. RNA blotswere analyzed with discriminating α³²P-dCTP-labeled probes generatedfrom the respective FcRH cDNAs. The following probes were used: (Top) aPCR-generated, 257-bp probe specific to the 3′ UT region of FcRH1;(Middle) a PCR-generated, 290-bp probe corresponding to the 3′ UT regionof FcRH2; and (Bottom) a 528-bp EcoRI-digested fragment of the 5′ end ofthe FcRH3 cDNA corresponding to its 5′ UT region, S1, S2, and EC1domains. The relative mRNA abundance was indicated by β-actin probe. Allthree FcRH gene probes hybridized with transcripts in the secondarylymphoid organs, spleen and lymph node. An FcRH1-specific probehybridized with spleen and lymph node transcripts of about 3.5 kb andabout 6.0 kb. Additional hybridization bands of about 0.7 kb and about1.5 kb were observed for heart, skeletal muscle, kidney, liver, and, inless abundance, placental tissue. Larger transcripts also were seen inskeletal muscle (about 6.0 kb) and in kidney and placenta (about 4.4kb). An FcRH2-specific probe hybridized to about 3.0-kb, about 4.4-kb,and about 5.5-kb transcripts most abundantly in spleen and lymph node. Atranscript of approximately 2.4-kb was notable in the kidney. An FcRH3probe hybridized with about 3.5-kb, about 5.5-kb, and about 7.0-kbtranscripts chiefly in spleen and lymph node. These also were seen,albeit in lesser abundance, in peripheral blood lymphocytes, thymus, andbone marrow samples. Additionally, a unique transcript of about 1.35 kbwas evident in skeletal muscle. These results indicated expression ofFcRH1, FcRH2, and FcRH3 in peripheral lymphoid organs, whereas tissuespecific differences in alternative splicing or polyadenylation weresuggested by the differential expression of transcripts with variablesize in nonlymphoid tissues. RTPCR analysis to date of non-lymphoidtissue skeletal muscle, however, does not reveal transcripts despite theNorthern analysis results.

When FcRH expression was examined by RT-PCR analysis of cell linesrepresenting different hematopoietic lineages, FcRH1, FcRH2, and FcRH3expression was found in every mature B cell line tested (Table 2). FcRH2and FcRH3 expression was limited to the mature B cell lines and not seenin the other types of cells examined. In contrast, FcRH1 expression wasseen in pro-B, T, and myeloid cell lines, although not in an erythroidcell line.

TABLE 2 Expression of FcRH transcripts in human B cell lines Cell TypeCell line FcRH1 FcRH2 FcRH3 Pro-B REH + − − Nalm 16 + − − Pre-B 697 − −− 207 − − − OB5 − − − B Ramos + + + Daudi + + + Raji + + + T Jurkat + −− Monocytic THP-1 + − − Myelomonocytic U937 + − − Promyelocytic HL-60 +− − Myelocytic KG-1 + − − Erythroid K562 − − − FcRH1, FcRH2, and FcRH3expression in cell lines was determined by RT-PCR.

RT-PCR analysis of sorted populations of peripheral blood cellsindicated that FcRH1, FcRH2, FcRH3, and FcRH5 are expressed atrelatively high levels in CD19+ B cells, whereas FcRH4 was expressed atonly trace levels. FcRH3 expression was observed in CD3+ T cells whereastranscripts of FcRH1 were barely detectable. FcRH1 expression also wasobserved in circulating granulocytes.

To refine the analysis of FcRH expression in secondary lymphoid tissues,tonsillar lymphocyte subpopulations were isolated. The five discretesubpopulations of B lineage cells, which can be distinguished by theirdifferential expression of cell surface IgD and CD38, representdifferent stages in B cell differentiation: follicular mantle(IgD+CD38), pre-GC (IgD+CD38+), GC (IgDCD38+), memory (IgDCD38), andmature plasma cells (CD38²+) (Pascual, V. (1994) J. Exp. Med. 180:329-339). RT-PCR analysis of FcRH1-5 expression in tonsillar B cellsubpopulations was performed. Viable cells were magnetically sorted intoCD19-non-B cells and CD19+B cells. The latter were stained with anti-IgDand anti-CD38 mAbs, and the five subpopulations indicated (CD38−IgD−,CD38−IgD+, CD38+IgD+, CD38+IgD−, and CD38²+) were sorted by flowcytometry. RT-PCR analysis of FcRH transcripts in non-B cells and the Bcell subpopulations was also performed. After cDNA preparation, PCRamplification was performed on the equivalent template of approximately10 k cells. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) wasamplified as a positive control.

RT-PCR analysis indicated little or no expression of FcRH transcripts inthe non-B lineage CD19-cells, most of which are T cells. However, CD19+subpopulations displayed coordinate expression of FcRH1, FcRH2, andFcRH3 transcripts in follicular mantle, naïve, GC, and memory B cellsubpopulations, but yielded no evidence of FcRH transcripts in pre-GC Bcells or plasma cells. In contrast, FcRH4 transcripts were restricted tothe follicular mantle and memory B cells, whereas FcRH5 expressionextended to mature plasma cells.

The relationship between the five FcRHs was examined by comparing theirfull-length, extracellular, and individual Ig-like domain amino acidsequences. This analysis, which included a recently identified mouseFcRH ortholog (moFcRH) and members of the FcR family, used the CLUSTALmethod algorithm (Higgins, D. G. & Sharp, P. M. (1989) Comput. Appl.Biosci. 5, 151-153). Comparison of the full-length sequences of otherFcRH family members with FcRH3 indicated 40-47% identity. By comparison,the degree of FcRH3 homology with the moFcRH was found to be 35% and21-24% with FcR members residing on chromosome 1, FcγRI, FcγRII,FcγRIII, and FcεR1. A lower level of amino acid identity (14%) wasobserved for the chromosome 19 LRC member, FcαR. A slightly higherdegree of extracellular homology was evident. Pairwise analysis of theindividual Ig-like subunits indicated conservation in membrane-distal tomembrane-proximal ordering of extracellular domain composition amongfamily members. Although similar Ig domain subunits were shared amongfamily members, the individual receptors were found to be composed ofunique domain combinations. The extracellular domain configuration ofthe moFcRH most closely resembled that of FcRH2, with which it has 46%identity. The extended pairwise comparison of the FcRH family with knownFcRs suggested the conservation of these Ig-like domains to some degreethroughout the greater family. The resemblance is particularly evidentin the FcRH3 membrane-distal domains that correspond to the three FcγRIdomains and the two domains of FcγRII, FcγRIII, and the FcR γ-chain.This analysis suggests the ancestral occurrence of differentialduplication and diversification of the individual Ig-like subunits inthe respective FcRH family members. The data also indicate that theFcRHs are more similar to their FcR neighbors on chromosome 1 than totheir FcR relative on chromosome 19.

The genomic sequence analysis of relevant chromosome 1q21 BAC clonesindicated that the entire FcRH locus spans 300 kb. The FcRH genes lie inthe same transcriptional orientation toward the centomere. Exon-intronboundaries were characterized by sequence comparison of their respectivecDNA clones and the AG/GT rule. The FcRH1 gene consists of 11 exons and10 introns spanning about 28 kb. The first exon, 5′ UT/S1, encodes the5′ UT region, the ATG translation initiation codon, and the first halfof a split signal peptide. S2, the second exon, is separated from 5′UT/S1 by a long intron of 12.9 kb and, like the neighboring FcRs, is 21bp in length (van de Winkel, J. G. & Capel, P. J. (1993) Immunol. Today14, 215-221; Kulczycki, A., Jr. et al. (1990) Proc. Natl. Acad. Sci. USA87, 2856-2860; Pang, J. et al. (1994) J. Immunol. 151, 6166-6174). Theextracellular region is encoded by three closely clustered exons,EC1-EC3, that code for the three Ig-like domains. The membrane-proximal,transmembrane, and the proximal portion of the cytoplasmic domain areencoded by a single sixth exon, TM. The cytoplasmic tail is encoded byfive exons, CY1-CY5, and the CY5 also encodes the beginning of the 3′ UTregion.

FcRH2 contains 12 exons and 11 introns that span 30 kb. It also containstwo exons that encode a split signal peptide, the first of which,5′UT/S1, includes the 5′ UT region, the ATG translation initiationcodon, and first half of the signal peptide. The second exon, S2, is 21bp in length. Exons 3-6 encode the four extracellular domains, EC1-EC4.The seventh exon encodes the membrane-proximal, transmembrane, and theproximal portion of the cytoplasmic domain. The FcRH2 cytoplasmic tailis encoded by five exons, CY1-CY5, the last exon of which includes thetermination of the ORF and beginning of the 3′ UT region.

The FcRH3 gene consists of 16 exons and 15 introns that span about 24kb. Unlike FcRH1 and FcRH2, its 5′ UT region is encoded by two exons, 5′UT1 and a second, 5′UT2/S1, that also encodes the ATG translationinitiation codon and the beginning of the split signal peptide. Thethird exon, S2, is also 21 bp in length. Extracellular domains encodedby six exons, EC1-EC6, are followed by exon 10 that encodes themembrane-proximal, transmembrane, and the proximal portion of thecytoplasmic domain. The cytoplasmic tail is encoded by five exons,CY1-CY5; the last contains the beginning of the 3′ UT region.

Example 2 Identification of HuFcRH6

FcRH6 is located in the midst of the classical FcRs at 1q21-23. Itsgenomic structure indicates, like the classical FcRs and FcRH1-5, asplit hydrophobic signal peptide encoded by two exons the second ofwhich is 21 bp.

FcRH6 was characterized using the methods described in Example 1. Acomposite analysis of Ig-like domains for relatedness with the otherhuFcRHs was performed. See Figure xxx. Sequence analysis of huFcRH6indicates its type I transmembrane form contains a consensus motif for asingle ITAM, or a single or two ITIM's.

Initial RT-PCR analysis of huFcRH6 in human tissues and cell lines (asdescribed in Example 1) reveals transcript expression in normal tonsiland lymph nodes. In cell lines, expression of huFcRH6 was identified inmyeloid cell lines THP-1 (monocytic), U937 (myelomonocytic), and KG-1(myelocytic). Limited expression if any was identified in the 207 pre-Bcell line and the Daudi B cell line.

Example 3 Generation of Transfectants and Antibodies

Recombinant constructs for transfection and stable expression ofhuFcRH1-5 have been generated. The constructs have been ligated into aCMV driven mammalian expression vector with and without greenfluorescent protein (GFP) fusion at the carboxyl terminus. Surfaceexpression of huFcRH1 and huFcRH3 was detected for both GFP and non-GFPforms by staining with antibody supernatant. The antibody supernatantwas derived from hybridomas generated by mice immunized with recombinantextracellular protein of the respective FcRH. The constructs forhuFcRH2, 4, and 5 have been detected by green fluorescence as well assurface expression for FcRH4.

Monoclonal antibodies have been generated, including, for example, anantibody that binds FcRH1. The preliminary analysis of FACS staining forFcRH1 expression with monoclonal antibody 1-5A3 labeled with a FITCconjugate (mouse anti human FcRH1) in peripheral blood from normalvolunteers indicates virtually all CD19+B cells have huFcRH1 expression,as do CD14+ monocytes and CD13+ granulocytes. CD3+ T cells have limitedto no expression of FcRH1. Staining of B-CLL samples from two differentpatient peripheral blood samples indicates that virtually allCD5+/CD19+B-CLL cells are positive for the FcRH1 1-5A3 antigen. Bywestern blot analysis of recombinant protein for FcRH1-5 extracellularregions 1-5A3 appears specific for FcRH1. 1-5A3 also stains B cell linesDaudi and Raji.

Example 4 Identification of MoFcRH1-3

A family of three mouse Fc Receptor Homologs (MoFCRHs) were identifiedand cloned. Amino acid sequences from the membrane proximal Ig-likedomains of huFcRH1-5 were used to identify putative mouse FcRH orthologsin the NCBI or Celera genomic, EST, and protein databases using theprotein BLAST (BLASTP) and the translated nucleotide BLAST (TBLASTN)algorithms, respectively. The location of moFcR family is split betweenchromosomes 1 and 3 in regions syntenic with human chromosome 1q21-23.See FIG. 4. The mo FcRH are located on mouse Ch3. Approximate positionswere determined from Genbank, Celera, and Mouse Genome Informaticsdatabases. Contigs of ESTs were generated to determine the putative cDNAsequences.

Genomic organization was determined by comparing cDNA clones generatedfrom RACE PCR with GenBank and Celera genomic sequences. DNASTAR™software (DNAStar, Madison, Wis.) was used for analysis of exon-intronboundaries which were characterized by sequence comparison and the AG/GTrule. All three genes contain a split signal sequence with a 21 bp S2exon (exon 2) which is found in all FcR and huFcRH genes on humanchromosone 1.

A comparison of tyrosine based motifs in FcRH cytoplasmic tailsindicated homology with the huFcRH family. See FIG. 5. An analysis ofsequence homology conservation is further shown in FIGS. 6 and 7.

Expression of the moFcRHs in tissue and cell lines was alsocharacterized as described in Example 1. Briefly, RT-PCR was performedon mouse tissues and cell lines with gene specific primers. Viabletissue was placed in TRIZOL® reagent (Molecular Research Center,Cinncinnati, Ohio) for RNA extraction. After cDNA preparation PCTamplification was performed on equivalent template amounts. Actin wasamplified as a positive control. McFcRH3 appears to have preferentialexpression in cells of B lineage. The results are shown in Tables 3-4.

TABLE 3 Tissue Distribution of moFcRH Expression TISSUE MoFcRH1 MoFcRH2MoFcRH3 Bone Marrow + + + Thymus + + + Spleen + + + Lymph Node + + +Peyer's Patches + + + Peripheral Blood + + + Brain + − − Liver + + −Heart + − − Muscle + − − Kidney + − − Lung + + − Intestine + + +Testes + − −

TABLE 4 Expression of moFcRH transcripts in cell lines Cell Type Cellline FcRH1 FcRH2 FcRH3 Pro-B SCID7 + +/− + Raw8.1 + + − Pre-B70Z/3 + + + BC76 − + + 18-81 + + + Imm. B WEHI-231 + + + WEHI-279 + + +B A20 + + + X16C8.5 + + + T EL4 + +/− −/+ NKT NKT + +/− − NKT 2C12 + +/−− Myeloid WEHI-3 + − − Lymphoid YAC-1 + + − Fibroblast 3T3 + +/− −Expression in cell lines was determined by RT-PCR.

The mouse FcReceptor Homologs include secreted or type I transmembraneisoforms that have unique cytoplasmic tails with potential activationand inhibition motifs. Their chromosomal location, Ig domain homology,and genomic organization indicate the mouse FcReceptor Homologs areorthologs of the huFcRH that have evolved a significant level ofdiversity. moFcRH1, moFcRH2, and moFcRH3 are predicted to encodesecreted or type I transmembrane proteins based on their amino acidsequences. moFcRH1 has two secreted isoforms both of which haveextracellular (EC) regions of four Ig-like domains with five potentialsites for N-linked glycosylation. One isoform is a fusion protein with atype B scavenger receptor domain containing 8 cysteines. moFcRH2 hassecreted and type I isoforms containing two Ig-like domains with fiveN-linked glycosylation sites. The type I isoform has an unchargedtransmembrane region which the secreted isoform lacks. Both isoformscontain the cytoplasmic portion which is long in the transmembrane formand contains five tyrosines including a consensus sequence for onepotential immunoreceptor tyrosine-based activating motif. moFcRH3contains five Ig-like domains with six potential sites of N-linkedglycosylation. Its transmembrane domain is also uncharged and thecytoplasmic region contains one potential ITAM and one potentialimmunoreceptor tyrosine-based inhibitory motif. The amino acid (aa)length of individual regions and full length (FL) isoforms, as well asapproximate molecular weight (MW) in Daltons (Da), is indicated in thestructural diagram of FIG. 8.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A purified antibody or antigen binding fragmentthereof, wherein the antibody or antigen binding fragment thereofselectively binds to a sequence of FcRH6, wherein the sequence consistsof SEQ ID NO:26.
 2. The antibody or fragment of claim 1, wherein theantibody or fragment is a monoclonal antibody or fragment thereof. 3.The antibody or fragment of claim 1, wherein the antibody or fragmentthereof is a humanized antibody, a fully human antibody, or a fragmentthereof.
 4. The antibody or fragment of claim 1, wherein the antibody orfragment thereof is a single chain antibody or fragment thereof.
 5. Theantibody or fragment of claim 1, wherein the antibody or fragmentthereof is labeled.
 6. The antibody or fragment of claim 1, wherein thelabel is a radiolabel.
 7. The antibody or fragment of claim 1, whereinthe antibody or fragment is conjugated or fused with a toxin.